XF Cell Mito Stress Test Kit User Manual
Contents
1. Antimycin A a complex III inhibitor binds to the Qi site of cytochrome c reductase thereby inhibiting the oxidation of ubiquinol in the electron transport chain of oxidative phosphorylation The inhibition of this reaction disrupts the formation of the proton gradient across the inner membrane Therefore the production of ATP is subsequently inhibited as protons are unable to flow through the ATP synthase complex in the absence of a proton gradient Mito Inhibitors A amp B COCR ATP Coupler ETC Accelerator Antimycin A Figure 1 1 Example of the Stress Oligomycin FCCP amp Rotenone Test 294 265 236 207 Capacity 178 149 4G 120 OCR pMoles min 62 33 itochondrial Respiration o 12 24 36 49 61 73 85 37 103 121 TIME min Basal respiration is predominantly controlled by the parallel re entry pathways through the ATP synthase and leak Addition of Oligomycin blocks the ATP synthase and the residual respiration is due to the proton leak The decrease on adding Oligomycin approximates to the proton current flowing through the ATP synthase before the inhibitor was added The decrease compared to basal provides the coupling efficiency The Seahorse Bioscience 2 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions addition of a carefully calibrated concentration of the protonophore FCCP introduces a high artificial proton conductance into the membrane This maximal re2. e y M9IAJ9AQ dnjes uoneziundo ej gt 2 S 3 lt N o H e 3 dnjes 1S9 SS9J1S sis jeuy 1S9 SS9J1S 5 Stress Test Setup This section covers the following topics e XF Cell Mito Stress Test Assay e Cell and Cartridge Preparation e Preparing Reagents e Setting Up the XF Cell Mito Stress Test Software XF Cell Mito Stress Test Assay Prior to Day of Assay Hydrate Cartidge and store overnight at 37 C Day of Assay Prepare Assay S Medium Stock 1 5 hours Seed Cells in XF Microplate Assumptions The optimal cell seeding density has been determined prior to running the optimization assay The average basal OCR was determined for the optimized cell seeding density The user has run the optimization assay for all 4 compounds for the cell line or cell type in question Seahorse Bioscience M9IAJ9AQ sisA jeuy dns sjuoBeoy uoneziundo uoneziundo sis jeuy 1S9J SS9J1S o EI o KA 7 o o Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Cell and Cartridge Preparation The day before running the assay do the following 1 Hydrate the desired number of cartridges and store at 37 C with no CO 2 Prepare the desired numer of cell plates by seeding cells at the appropriate density Preparing Reagents 1 Determine the appropriate concentration of each injected compound by running the compound optimization experiments and
3. The dominant pathway of proton reentry is via ATP synthase In the absence of a proton motive force the synthase acts as a proton pump hydrolyzing matrix ATP and extruding protons from the matrix However the high proton motive force generated by the electron transport chain forces the complex to run in reverse synthesizing matrix ATP as protons are driven back through the complex The electron transport chain proton pumps together with the ATP synthase thus creating microscopic proton circuits across the inner mitochondrial membrane The circuit has terms of potential the proton motive force in millivolts and flux the proton current in nanomoles of protons per minute This proton circuit is central to mitochondrial bioenergetics investigations Experimentally distinct but parallel experimental approaches are used to quantify the potential and flux components of the proton circuit both with isolated mitochondria and intact cells The proton motive force is expressed in millivolts and is identical to the proton electrochemical potential which uses the thermodynamic unit of kJ mole A useful conceptual model to consider is the proton circuit being analogous to a simple electrical circuit Figure 6 7 An additional component is necessary to provide a more accurate model of mitochondrial bioenergetic function All mitochondria possess an endogenous proton leak that is apparent also in mitochondria in situ within intact cells and is thus not an artif
4. eese 3 Comp rids ceto ot rob tr reine 5 MEDI 3 Preparing iue iater edd 6 Red top vial eese 5 Hequirernnts ie corre ro ea 3 Resuspending Compounde eser lenne 6 Seahorse Bioscience S Safety cabinet eese 3 Seahorse assay medium 3 9 Serial dilution vessel eese 3 Setting up optimization software 10 Setting up sterss test software 21 Spare respiratory capacity eiee ii 27 Standard cell culture equipment 3 Stress test ASSUIMPUIONS 3 2 5 253 5 xix grana vion inue ades eege 19 Data Gresentation cii 26 Preparing cartridge for injection and cell plate 20 Preparing reagente i 20 Stress Test Kit el 3 Example edet oen ite esito 2 OVEIVIOW 1 Support Technical iv T Technical support eese iv Text conventions eese iii U URBE uiu nibus uiid iv V Viele m M 3 Ww Website support iv X XF excel KU 25 XF24 Extracellular Flux Analyzer 3 MELISDg g 3 Y Yellow top Vial coeno eorr ain 5 36 Seahorse BioScience XF Cell Mito Stress Test Kit XF24 Instructions Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit XF24 Instructions This page intentionally left blank Se
5. ASSAY TEMPI ATELO 24 al EJ 7 XF Mito Stress Test Kit a ES Figure 5 4 Stress Test j P p T Injection Layout Screen Stress Test Injection Layout ZA o O ae o m o __ ATP Coupler 0 50UM ETC Accelerator 0 501 Evito inhibitor 0 50um O 3 c N Ue 9 Click Start 2 10 Choose a directory to save the file in change the default file name if necessary and then click OK 11 Place the cartridge and calibration plate with loaded injector ports on the slide out tray O 12 When prompted replace the calibration utility plate with the cell plate gt 2 53 13 Click Start lt N vo my red 14 The Stress Test will now run on the XF Analyzer 15 When the run is over follow the prompts in the software and remove the cartridge and cell plate and discard o EI o o 7 o o o KE 590 D O e i o o P Seahorse Bioscience e Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions This page intentionally left blank 7 EI oO I 7 4 oO kd Seahorse Bioscience 6 Stress Test Analysis This section covers the following topics Data Presentation Spare Respiratory Capacity Coupling Efficiency Basal Respiration ETC Accelerator Response ATP Coupler Response The Mitochondrial Proton Circuit e Mitochondrial Proton Current Respiration Since mitochondrial function can be analyzed in a number of different ways one of the key
6. Average basal OCR 7 11 19 22 B Basal Fesplratiori ceni certet 2 29 Hesponse ee seeeennnnnneeanannnnnetao 27 Bench top Mini centrifuge u eee eeeeeeseneeeeenete 3 VOMOX em 3 Blue top vial neret iter tete dpt 5 C Cartridge preparation Optimization eee eeeeeeeeeeeeeeeneeeeseeeeeneeeesaees 8 Stress test EE 20 Cell culture incubator AN 3 Cell preparation Optimization eee eeeeeeeeeeeeeeeneeeeeeeeeeeneeeesaees 8 Stress EE 20 Cellular metabolism esses 1 ecbreTe 3 Chemincal names a 3 Class II Biological Safety Cabinet 3 6 Coenzyme Qu eere deg tertie reveren 2 Complex Rule el 2 Ri lee 2 Massen eti peak aid sta ne AR ER ap ida axe eR RR ERR REFERS 1 Seahorse Bioscience Compounds resuspendng ss iii 6 Contact information eese iv Viel le EE iii Coupling efficienCy 27 28 D Determining Inhibitory concentration 17 Peak response for ETC accelerator 14 E ETC Accelerator esee 3 5 27 R6SpOFfISO nei reti een 27 30 Extracellular Acidification Rate ECAR 1 G Getting Ee reete titer teer tanen iv Green top vial inner teneris 5 Grey top Vial er inire eor ceteras 5 H Hardware reouirements reese
7. Cell Mito Stress Test Kit User Manual XF24 Instructions An important diagnostic of the bioenergetic status of a cell particularly one which can experience a variable ATP demand such as a neuron is to determine the spare respiratory capacity the ability of substrate supply and electron transport to respond to an increase in energy demand Ideally this should be done by increasing oxidative phosphorylation directly by activating an extra mitochondrial ATPase but since this is not always possible an alternative is to add a carefully titrated concentration of protonophore such as FCCP sufficient to induce uncontrolled respiration Under these conditions the mitochondria can still retain a considerable proton motive force and are still able to generate ATP The FCCP concentration is critical since excess will collapse the proton motive force Coupling Efficiency The coupling efficiency metric measures the amount of ATP turnover in the mitochondria compared to a baseline reading The coupling efficiency data can be found by scrolling down to below the spare respiratory capacity data in the Mito Stress Test Output tab Figure 6 3 Coupling Average Efficiency 83 896 65 696 71 6 39 D presented as a Be response 62 3 66 0 E from basal readings with Coupling Efficiency non mitochondrial respiration subtracted out These data are In a single experiment it i
8. This compound is the most sensitive in the kit to concentration changes so it is important that the correct peak data point is chosen from the optimization experiment 1 When the optimization experiment is complete open the excel data output file and choose the worksheet tab labeled Mito Stress Test Optimization see Figure 4 1 ETC Accelerator OCR pMoles min e 010 198 00 Figure 4 1 938 oencentration at Peak Screen shot of ul to add to the Mito Stress Test me Optimization stock excel output for the ETC Accelerator ETC Accelerator Dose Response E x 9 0 10 1 00 ETC Accelerator uM 5 nat Data Viewer Mito Stress Test Optimization Assay Configuration Level Rate Data Rate Data Pate View TINN The graph on the left shows a dose response for the compound tested The table in the middle shows the data used to generate the graph compound concentration and OCR response The software will calculate the peak response from the graph in the green box labeled Concentration at Peak The user can change this concentration as needed to determine the proper volume of compound stock to add to 5ml Seahorse Assay Medium for the Cell Mito Stress Test 2 The software automatically produces a dose response curve for the compound tested This curve is shown with compound concentration on the x axis and OCR on the y axis 3 The software will automatic
9. cells which is defined as the quantitative difference between maximal uncontrolled OCR and the initial basal OCR It has been proposed that the maintenance of some spare respiratory capacity even under conditions of maximal physiological or pathophysiological stimulus is a major factor defining the vitality and or survival of the cells The ability of cells to respond to stress under conditions of increased energy demand is influenced by the bioenergetic capacity of mitochondria This bioenergetic capacity is determined by several factors including the ability of the cell to deliver substrate to the mitochondria and the functional capacity of the enzymes involved in electron transport The third injection is a combination of Rotenone Mito Inhibitor B a Complex inhibitor and Antimycin A Mito Inhibitor A a Complex III inhibitor This combination shuts down mitochondrial respiration and enables both the mitochondrial and non mitochondrial fractions contributing to respiration to be calculated A decrease in OCR due to impaired mitochondrial function will occur with a concomitant increase in ECAR as the cell shifts to a more glycolytic state in order to maintain its energy balance Rotenone is a mitochondrial inhibitor that prevents the transfer of electrons from the Fe S center in Complex I to ubiquinone Coenzyme Q This inhibition of Complex prevents the potential energy in NADH from being converted to usable energy in the form of ATP
10. injection The basal reading is then used to baseline the responses to different compounds o Figure 6 4 Basal Averepe lt Respiration 267 1 100 These data are se presented an 272 7 2 8 i 2558 139 x absolute OCR o9 274 8 25 6 reading pMoles min 073 Basal Respiration taken from the E N third basal He o measurement 3 ziundo SISAJEUY z uone Data Viewer Mito Stress Test Output Assay Configuration Levels Rate Data Ra dnies 1S9 SS941S SISAJEUY 7 Ki oO n 0 in Kd KI Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions ETC Accelerator Response The ETC Accelerator Response represents the maximum OCR measurement value after the ETC Accelerator FCCP injection It is calculated by taking the maximum value from the 3 after injection measurements This value is then used to calculate Spare Respiratory Capacity represented here to provide the user an idea of maximum OCR readings seen in the assay Average 635 2 ETC Accelerator Response pMoles min M M Data Viewer Mito Stress Test Output Assay Configurabon Levels Seahorse Bioscience Figure 6 5 ETC Accelerator Response These data are presented an absolute OCR reading pMoles min taken from the maximum OCR measurement after the ETC Accelerator injection 30 M9IAI9AQ dnies sisAjeuy dn
11. the peak is reached In this example the user should choose the concentration where the curve flattens out in this case 1 uM OCR pMoles min 0 01 0 10 1 00 10 00 ETC Accelerator uM Seahorse Bioscience i Maan s u e y dnies uoneziundo ej gt 2 S 3 lt N o os 3 dnjes 1S9 SS2J1S sis jeuy 1S9 SS2J1S Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions ETC Accelerator Dose Response o Figure 4 4 In this example the curve shows a steep increase in OCR over 450 a small range of concentrations Even though there is a higher peak s 400 point at 0 75 uM the user should 350 choose the point after the steep increase in this case 0 5 uM The e 300 idea is to maximize the response in t the lowest concentration possible 250 J amp 200 D S 8 150 o m 100 o 50 0 0 01 0 10 1 00 10 00 ETC Accelerator uM o wi o z NOTE When optimizing with the XF24 there is an inherent limit in the number of concentrations 2 Si that can be tested compared to the XF96 on the same plate For this reason if there is a steep 9 increase in the early or late parts of the curve it would be beneficial to re optimize with finer compound concentrations using the standard software gt 5 lt o o uoneziundo dnjes 1S9 SSO11S sis jeuy 1S9J SS9J1S Seahorse Bioscience is Seahorse BioScience XF Cell Mito Stress Test Kit U
12. 4 Instructions This page intentionally left blank Seahorse Bioscience 4 2 Reagent Preparation This section covers the following topics e Preparing the Reagents Aliquoting Reagents Reagent Kit Contents Figure 2 1 Kit Contents One box of reagent compounds contains enough material for 6 full XF24 assays The reagent box contains the following items e 4 vials of the reagent compounds in powder form marked with a Seahorse logo Yellow top vial ATP Coupler Blue top vial ETC Accelerator Red top vial Mito Inhibitor A e Green top vial Mito Inhibitor B e Grey top vial 1 ml DMSO used to resuspend the compounds e 20 pre labeled vials for aliquoting Seahorse Bioscience M9IAJ9AQ dnies uoneziundo SISAJEUY UONEZIURA O dni9s 1S9 SS941S SISAJEUY 189 SS9J1S poj oO D ite oO gt m 7 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Preparing the Reagents This protocol describes the process of resuspending the compounds in the appropriate volume of DMSO to give a 2 5 mM stock concentration for each compound 1 Remove the XF Cell Mito Stress Test Kit from the 20 C freezer and place on a lab bench for 30 minutes to thaw NOTE When handling the compounds chemical resistant impervious gloves should be worn 2 Oncethe reagent compounds are resuspended and aliquotated they must be used within 6 weeks when stored at 20 C
13. Mitochondrial Proton Current Respiration esessesseseeeneeeenneneen nnne 33 WI EE a a SE Seahorse Bioscience vi Overview of the Manual Section 1 Overview of XF Cell Mito Stress Test Kit on page 1 contains an overview describing the scientific merits of the stress test and a description of the kit contents and other required items Section 2 Reagent Preparation on page 5 describes how to prepare and aliquot the reagents Section 3 Optimization Setup on page 7 describes how to set up the optimization assay and software e Section 4 Optimization Analysis on page 13 describes how to analyze the results of the optimization assay e Section 5 Stress Test Setup on page 19 describes how to set up the XF cell mito stress test assay and software Section 6 Stress Test Analysis on page 25 describes how to analyze the results of the XF cell mito stress test assay Seahorse Bioscience ii Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions This page intentionally left blank Seahorse Bioscience vi Overview of XF Cell Mito Stress Test Kit This section covers the following topics e Overview e XF Cell Mito Stress Test Kit Contents e Other Requirements Overview The ability to measure cellular metabolism and understand mitochondrial dysfunction has enabled scientists worldwide to advance their research in understanding the role of mitochondrial f
14. User Manual XF cell mito stress test kit for research use only XF24 Instructions Seahorse Bioscience www seahorse Part 101848 400 Rev C This page intentionally left blank Preface Copyright 2010 Seahorse Bioscience Inc All rights reserved Printed in U S A Under copyright laws this manual may not be reproduced in any form in whole or in part without prior written permission from Seahorse Bioscience Inc This revision supercedes all previous revisions Every effort has been made to ensure that the information in this manual is accurate at the time of printing However Seahorse Bioscience Inc assumes no liability for errors or omissions and reserves the right to make changes without notice to any products described herein to improve reliability function or design Excel is a registered trademark of Microsoft Corporation Other company and product names may be trademarks of their respective companies Conventions This guide uses the following conventions Convention Type of Information Bold Indicates user interaction with elements of the software or system Titles are spelled as they appear on the system 1 Procedures are numbered and subprocedures are lettered You must complete steps in the sequence they are presented to ensure 2 success a Bulleted lists indicate general information about a procedure They do not imply a sequential procedure Information HINT Provides a hel
15. ZA oO D e br 3 7 3 Ina Class Il Biological Safety Cabinet resuspend powder reagent compound marked with the Seahorse logo in 180 ul of DMSO Vortex the vial right side up and up side down for 10 seconds each Spin down the vial in a mini centrifuge for approximately 5 seconds Repeat steps 1 through 4 to resuspend the other reagent compounds xn 9 n R Write the date of resuspension in the Date Reconstituted box on the side of the XF Cell Mito Stress Test Kit Aliquoting Reagents 1 Openall the empty vials of the same color for one resuspended reagent for example all of the red toped vials 2 Using a P 200 pipette aspirate 30 ul of the resuspended reagent in this example the red vial containing the Mito Inhibitor A reagent 3 Dispense 30 ul of reconstituted reagent into the appropriate empty vial capping the vial immediately 4 Repeat steps 1 through 3 for each resuspend reagent until each tube contains 30 ul of reagent 5 Reagents that are not used immediately should be stored in the XF Cell Mito Stress Test Kit box at 20 C NOTE Once the reagent compounds are resuspended and aliquotated they must be used within 6 weeks when stored at 20 C Seahorse Bioscience 3 Optimization Setup This section covers the following topics e Optimization Assay e Cell and Cartridge Preparation e Optimization Assay Example for XF24 e Setting Up the Optimization Software Optimizati
16. act of isolation In the absence of ATP synthesis Seahorse Bioscience sa SISAJEUY 7 EI O 7 4 oO o Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions the proton circuit is largely completed by the proton leak which may serve as an important factor in limiting the proton motive force therefore restricting leakage of single electrons from the electron transport chain to form the superoxide anion The ATP synthase and the proton leak provide parallel pathways for proton reentry The electrical analogue can be quantified in terms of voltage current and by Ohm s Law resistance or conductance In the proton circuit the corresponding parameters are proton current nmol H min per unit of mitochondrial protein or cell number proton motive force in mV and proton conductance nmol H min mV per unit material Mitochondrial Proton Current Respiration The tight coupling between electron transport and proton extrusion attests that for a given substrate the rate of mitochondrial oxygen utilization with both the isolated organelles and intact cells is an accurate measure of the total proton current The many techniques available for monitoring the rate of oxygen utilization by mitochondria all measure the activity of a single process which is the transfer within complex IV of 4 electrons to a molecule of oxygen to generate two molecules of water Despite this oxygen electrode experiment
17. advantages to using the XF Cell Mito Stress Test Kit is that key metrics are calculated automatically by the software allowing for easy standardization between runs Upon completion of the XF Cell Mito Stress Test the data outputs to a standard XF excel file The standard analysis tabs are still present Data Viewer Rate Data etc as well as the troubleshooting tabs that contain the raw data Levels Calibration When the XF Cell Mito Stress Test is run an additional tab is present called the Mito Stress Test Output Click on this tab to view the data calculated from this experiment SISAJEUY 7 EI O D 4 o o Seahorse Bioscience e Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Data Presentation Data from five separate metrics are automatically calculated by the XF Cell Mito Stress Test software and presented in the format shown in Figure 6 1 G XF24 Stress T A 8 Mito Stress Test Output Figure 6 1 Example data Spare Respiratory Capacity Average StdDev output for the 1 2 3 4 5 14 o 8 d 2 D 257 3 10 6 6 XF Cell Mito 6 5 159 96 7 8 153 196 8 896 195 356 9 1 e Stress Test 308 296 14 596 171 296 21 396 237 0 13 3 Spare Respiratory Capacity Data Viewer Mito Stress Test Output Assay Configuration Levels RateData Rate li Data Table The data table in the upper l
18. ahorse Bioscience Corporate Headquarters European Headquarters 16 Esquire Road Symbion Science Park Boks 22 North Billerica MA 01862 US Fruebjergvej 3 Phone 1 978 671 1600 800 671 0633 2100 Copenhagen DK Seahorse Bioscience
19. ally suggest a concentration at the peak of the curve but it is very important for the user to check that this is the true peak and change accordingly 4 When the peak concentration is determined type it into the field labeled Concentration at Peak Seahorse Bioscience is M9IAJ9AQ s u e y dnies uoneziundo gt 5 I lt 4 o dnies 1S9 SS941S SISAJEUY 189 SS9J1S uoneziundo Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions 5 The software will automatically calculate the volume of 2 5 mM compound needed to make 5 ml of the final injection the red box Write this volume down in your notebook and use this to prepare injections for the cell mito stress test Determining the Peak In some situations the peak response is not always obvious The following presents a few examples of data from an optimization experiment and how Seahorse recommends the peak concentration be chosen ETC Accelerator Dose Response Figure 4 2 In this example the answer if obvious the OCR values 450 rise as ETC Accelerator concentration increases and falls 400 after the peak is reached Here the 350 user should choose 0 75 uM as the optimized concentration e 300 E i 250 amp 200 x 8 150 100 50 0 0 01 0 10 1 00 10 00 ETC Accelerator uM ETC Accelerator Dose Response Figure 4 3 In some cases the OCR response rises gently with concentration and levels out once
20. analyzing the date 2 Prepare 5 ml of each compound to be injected The Mito Inhibitor A and Mito Inhibitor B are added at the same time during the assay so the user should prepare 1 5 ml injection stock with both compounds added The concentration of each stock compound is 2 5 mM The volume of the 2 5 mM stock to be used for preparing the 10x injection compound is automatically calculated in the Optimization tab If calculating manually prepare a 10X injection concentration compared to the final desired working concentration NOTE Seahorse recommends preparing 5 ml of each injection so the user can use a multichannel pipette reservoir The actual volume needed to fill the 20 ports for injection is 1 5 ml If the optimized concentration is too high to make the full 5 ml recalculate to make 2 ml of injection stock Preparing Cartridge for Injection and Cell Plate 1 Add the appropriate volume of the prepared compound reagents into the appropriate injection port Port A 55 ul ATP Coupler e Port B 61 ul ETC Accelerator e Port C 68 ul Mito Inhibitor A and Mito Inhibitor B 7 EI oO I o oO 77 2 Store cartridge in a 37 C incubator with no CO until ready to use 3 Perform a medium change on the cell plate by removing the running medium from each well and replacing with Seahorse Assay Medium described in detail in the XF24 Extracellular Flux Analyzer User Manual Final vol
21. atnm tenete 8 Optimization Assay Example for SEA 8 Setting Up the Optimization Software E 10 4 Optimization Analysis Analyzing the Peak Response for the ETC Accelerator seen 13 Determining WEE 14 Analyzing the Inhibitory Response for the ATP Coupler and Mito Inhibitor A and Mito Inhibitor B 16 Determining the Inhibitory Concentration mene 17 5 Stress Test Setup XF Cell Mito Stress Test EE 19 ASSUMPTIONS EE 19 Cell and Cartridge Preparation tat rn nnne nnn etta tana i a cata i Ea I a Ru Rana 20 Preparing Razer naa 20 Preparing Cartridge for Injection and Cell Plate AAA 20 Setting Up the XF Cell Mito Stress Test Software 21 Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions 6 Stress Test Analysis Data SE te rn fesas IRE REL ERR ERR IHRE ELA RE LVXNT EIN A REM AMA XR ER RR RE deka ruk rakia bias 26 Spare Respiratory Capacity ccsececeseeceesceesesseneeseceeseseeeeeseeseaseneeseseeenseaseneseeeessenenaseeseneeeess 27 C upling une ne P 28 Basal Resplr tion 1e iret ENEE itinera ase t ru kept steve rane dea vo Ka nte di Dunn a o de 29 ETC Accelerator Response eeesesieesessseseeeeeeeee einen nennen nnns nnt enn nna denter nasi nnne nnn innen 30 ATP Go pler e 31 The Mitochondrial Proton Circuit sssrinin ai era Ear EST i 31
22. d choose 0 75 uM as the optimized concentration 250 a 8 o o OCR pMoles min o o 50 0 01 0 10 ATP Coupler uM 1 00 10 00 NOTE In this example the curve trends subtly downward once the curve bottoms out Here it is important that the user manually enter the 0 75 uM concentration in the green box in the spreadsheet as the software may automatically recommend a higher concentration Seahorse Bioscience i Maan s u e y dnies uoneziundo gt 5 lt o o dnjes 1S9 SSO11S sis jeuy 1S9J SS9J1S uoneziundo Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Mito Inhibitor A Dose Response Figure 4 7 In this example forMito Inhibitor A the curve shows a steep decrease in OCR over a small range of concentrations Here the proper concentration for the best inhibitory response is 0 5 uM 250 a 8 laj S OCR pMoles min o o 50 9 10 Mito Inhibitor A uM 90 10 90 Mito Inhibitor B Dose Response Figure 4 8 In some cases like this one for Mito Inhibitor B the curve shows a gradual decline in OCR over increasing concentrations Again the concentration where the curve flattens out at it s nadir is the 250 200 optimized inhibitory concentration A In this example that concentration 150 is 1 5 uM v z a 100 50 0 01 0 10 Mito Inhibitor B um 1 90 10 00 18 Seahorse Bioscience s u
23. e Complex II of which succinate dehydrogenase is a part is energetically incapable of pumping protons The stoichiometry of the proton pumps is such that a total of 10 protons are extruded for each electron pair passing from NADH to oxygen Therefore the rate of oxygen uptake by a cell after correcting for any non mitochondrial respiration is the indicator allowing measurement of the proton current The redox span across the electron transport chain is approximately 1100 mV and provides an estimate of the maximal proton motive force across the inner membrane vary from 180 mV to 220 mV Seahorse Bioscience si SISAJEUY 7 OD RK 7 o 7 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions V Ay or strictly p PIETA JH dO dt X H O The electrical circuit analogy of the proton circuit inner mitochondrial membrane pyruvate In Figure 6 7 Left the three proton pumping complexes of the electron transport chain a pump protons out of the mitochondrion generating a proton motive force The proton circuit is completed by re entry through the ATP synthase b coupled to the generation of ATP or through a dissipative leak c Right In the electrical analogy three electrical batteries corresponding to Complexes Ill and IV generate a voltage and the electron circuit is completed by useful work lighting a bulb or dissipative work through the variable resistance
24. eft hand portion of the screen shows the calculated metric for each individual well A user can scan the data quickly and note if there are any outliers in individual groups The groups previously chosen in the Stress Test Groups Layout screen within the Stress Test wizard are delineated by color in this table Note that the color code is consistent throughout the results data including the histogram Statistics Table The statistics table in the upper right hand of the screen shows average standard deviation and coefficient of variation CV for each individual group This provides the user an indication of the reproducibility between the groups Histogram The graph on the bottom of the screen shows a histogram with the metric of interest on the y axis and the group on the x axis This graph provides the user a snapshot view of the group data as they relate to each other SISAJEUY 7 OD RK 7 o 7 Seahorse Bioscience e Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions The data for the five output metrics are calculated according to Table 6 1 Table 6 1 Description or formula for each output metric explaining calculation Metric Description Formula Spare Respiratory Capacity ETC Accelerator Response Basal Respiration Coupling Efficiency 1 ATP Coupler Response Basal Respiration Basal Response The 3rd basal measurement measurement before injection ETC Accelera
25. ion Injection Concentration Final Working Seahorse Assay Volume UM 10X final working Concentration Medium ul from concentration LM previous well tube 1 30 3 1000 2 15 1 5 500 500 3 10 1 333 667 4 7 5 0 75 250 750 5 5 0 5 333 667 6 1 0 1 800 200 Refer to step 1a Preparing Cartridge and Cell Plate for Assay 1 See the plate map in Table 3 2 and dispense 75 yl of injection medium with compound into port A of the appropriate cartridge Figure 3 1 Well Plate Ports Table 3 2 24 Well Final Working Concentration 6 Blank 0 5 0 75 1 1 5 3 JM O 1 0 5 0 75 Blank 1 5 3 CG 0 1 0 5 Blank 1 1 5 3 DES 0 1 0 5 0 75 1 1 5 Blank 2 Place the cartridge with injections in a 37 C incubator with no COo until the assay is started 3 Perform a medium change on the cell plate by removing the running medium from each well and replacing with Seahorse Assay Medium described in detail in the XF24 Extracellular Flux Analyzer User Manual Final volume for each well is 675 ul 4 Place the cell plate in a 37 C incubator with no CO for one hour prior to the assay Seahorse Bioscience M9IAJ9AQ s u e y s s jeuy uoneziundo dnies 1S9 SS941S SISAJEUY 189 SS9J1S O c Li N ES ct o 5 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions NOTE Seahorse recommends medium changes using the XF Prep Station to insure accurate final volumes When using the XF Prep Stati
26. jes sjuebeey uoneziundo uoneziundo 1S9 egene SISAJEUY 7 Ki oO n 0 in Kd KI Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions ATP Coupler Response The ATP Coupler Response represents the minimum OCR measurement value after the ATP Coupler Oligomycin injection It is calculated by taking the minimum value from the 3 measurements after injection This value is then used to calculate Coupling Efficiency Figure 6 6 ATP Coupler Average StdDev Response 39 7 P 92 0 These data are et s presented an 980 x absolute OCR 93 0 9 reading pMoles min ATP Coupler Response taken from the minimum OCR measurement after the ETC Accelerator injection e PMoles mig D S G1 G2 G3 1 amp 7 M M Data Viewer Mito Stress Test Output Assay Configuration Levels Rate Data The Mitochondrial Proton Circuit The mitochondrial proton circuit Figure 6 7 is central to the multiple physiological functions of mitochondria Three electron transport complexes 1 Ill and IV pump protons across the inner mitochondrial membrane In each case a decline in redox potential of the electrons passing through the complex is coupled to the extrusion of protons from the matrix Complexes and Ill normally operate close to thermodynamic equilibrium and can be induced to reverse under artificial conditions Complex IV is irreversibl
27. n 3 so RTI T iv Hint statements eese iii leie tee 3 Inhibitory response analysis 16 Items YOU need eee ne 3 K Kit Contents cuir nere sedente etras nett uas 3 M Mini centifuge esee 3 Mito Inhibitor A netten 2 3 5 6 Inlilbitor Biete hens 2 5 Stress Test Output tab 25 28 Mitochondrial Dysf nctiOn io itte 1 FUNCTION 25 35 Seahorse BioScience XF Cell Mito Stress Test Kit XF24 Instructions Proton circuit eene 31 Proton current respiration 33 N Names Chemicals c3 t onte iara buka kintan kav eia 3 Fleagents ugereest SE eece urine 3 Note statements iii O OCH Detten eicere reae 1 Elle ele 1 Optimization PASS AY auct UN EU e LS UN 7 Assay example seeseeeeeenen 8 ASSUMPTIONS a ertet dna ta ea nsa un sonda Dc 7 Creating serial dilution 8 Preparaing cartridge for assay 9 Preparaing cell for assay 9 Preparing compoundS ete iinne e 8 Oxygen Consumption Rate OCR 1 P P10 P1000 pipettes AA 3 Peak response analysis tesenn 13 Phone number eese iv Preparing Cartridge rtr nce 8 20 GOI m M s 8 20 Ri Le gebeten PEU 6 R Reagents Chemical names
28. nsity Optimization Assay Example for XF24 For this example we will use a 24 well plate to do the dilutions You can also use eppendorf tubes Preparing Compounds for Injection Ports 1 Remove the reagent box from the 20 C freezer 2 Remove one tube for the reagent to be optimized and place the reagent box back in the freezer Creating a Serial Dilution NOTE To determine the volumes for the following procedure use Table 3 1 Serial Dilution Volumes 1 Perform serial dilution in a 24 well plate a Prepare 30 uM top concentration by adding 12 ul from the appropriate reagent vial to 988 ul of Seahorse running media into well 1 b Dispense the appropriate volume of Seahorse running media into wells 2 through 6 found in the Seahorse Assay Medium column in Table 3 1 c Using the pipette mix up and down in well 1 several times d Remove the appropriate volume of solution from well 1 found in the Dilution Volume column in Table 3 1 and add to well 2 For example for well 2 dilution remove 500 ul from well 1 and add to well 2 For well 3 dilution remove 667yl from well 2 and add to well 3 e Using the pipette mix up and down in well 2 several times Seahorse Bioscience O c Li N D ct o 5 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions f Repeat steps c through d until all 6 wells have solution Table 3 1 Serial Dilution Volumes Dilut
29. o SS B3 NOTE The average basal OCR value for the cell in question should have been determined prior to N running the optimization assay when optimizing for cell seeding concentration a 5 6 Click the Start 7 Choose a directory to save the file in change the default file name if necessary and then click OK 8 Place the cartridge and calibration plate with loaded injector ports on the sliding tray 9 Click Continue to start calibration dnjes 1S9 SSO11S 10 When prompted replace the calibration utility plate with the cell plate 11 Click Start 12 The Optimization Assay will now run on the XF Analyzer e e o 13 When the run is over follow the prompts in the software and remove the cartridge and cell plate and E 5 discard D Y lt 9 o o m Seahorse Bioscience y Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions O ke c Li N D ct o 5 This page intentionally left blank Seahorse Bioscience i 4 Optimization Analysis This section covers the following topics Analyzing the Peak Response for the ETC Accelerator e Determining the Peak Analyzing the Inhibitory Response for the ATP Coupler and Mito Inhibitor A and Mito Inhibitor B e Determining the Inhibitory Concentration Analyzing the Peak Response for the ETC Accelerator When the ETC Accelerator is injected into the wells the OCR response should increase in a dose dependent manner
30. on the final volume should be set to 675 ul Setting Up the Optimization Software 1 Open the XF software A XF Software Figure 3 3 Seahorse Mito Kit Screen Standard Seahorse Apps XF Cell Stress Test Kit v Start App About this screen 2 ln the Seahorse Apps drop down menu choose XF Cell Mito Stress Test Kit 3 Click the Start App button 4 Click the Run Optimization plate button Seahorse Bioscience L M9IAJ9AQ s u e y s s jeuy uoneziundo dnjes 1S9 SSO11S sis jeuy 1S9J SS9J1S e UO Eh 3 N ES e 5 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions The Optimization Injection Layout screen appears o Guest Seahorse X74 Current ale r MTOK OPTIMIZATION TEMPLATE A XT o K e ES XF Cell Mito Stress Test Kit KA Z Figure 3 4 Optimization FF oo Assay ETC Accelerator s Optimization Injection Layout Injection Map OPTIMIZATION A ETC Accelerator Cell seeding EE Average basal OCR 25 1000 Load Port A ae gt AU S D e 3 t 3 m o E JErc Accelerator Media Oniy 5 Onthe injection map screen do the following o 3 N ES ct o 5 a Choose the compound you are optimizing from the drop down menu at the top of the page b Inthe Cell seeding box enter the number of cells seeded per well c Inthe Average basal OCR box enter the average basal OCR for the seeded cell density
31. on Assay The optimized working concentrations of the compounds used in the XF Cell Mito Stress Test Kit can change based on the cell line or cell type used for the assay The Optimization Assay should be run for each compound when a new cell line is used Before running the optimization assay the user should run a cell titration assay described in the XF24 Extracellular Flux Analyzer Users Manual to determine the proper cell seeding density for each cell line or cell type Record the average basal OCR value for this cell density as it will be used to define the protocol in the software wizard Prior to Day of Assay Day of Assay Hydrate Cartidge and store overnight at 37 C E Prepare Assay Medium Stock 1 5 hours e Seed Cells in XF Microplate Assumptions The optimal cell seeding density has been determined prior to running the optimization assay The average basal OCR was determined for the optimized cell seeding density Seahorse Bioscience 7 M IM AO s u e y s s jeuy uoneziundo dnjes 1S9 SSO11S sis jeuy 1S9J SS9J1S e UO Eh 3 N ES e 5 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Cell and Cartridge Preparation The day before running the assay do the following 1 Hydrate the desired number of cartridges and store at 37 C with no CO 2 Prepare the desired number of cell plates by seeding cells at the appropriate de
32. pful hint related to the current topic NOTE Calls out a specific area of note in the protocol Seahorse Bioscience i Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Related Documentation In addition to the user manual a Quick Start Guide is shipped with each kit Customer Support To contact Seahorse Customer Service e By phone 800 671 0633 Option 3 e By fax 978 671 1611 By e mail support seahorsebio com e By web http www seahorsebio com By mail Seahorse Bioscience Inc 16 Esquire Road N Billerica MA 01862 Seahorse Bioscience Table of Contents 1 Overview of XF Cell Mito Stress Test Kit le IK EeE 1 XF Cell Mito Stress Test Kit Contents ssssssssssssccssssssssssssssssssssssesrsnssssnssssssssssttsss 3 Other Re ulleMents 3 2 Reagent Preparation Reagent Kit Contents zai ser o dias senpa sin ne ERE ER ERE TARA EUREN Ra EE Ea EUR ARZA IARE REA RE AS EN Kai EAATAT don NREEER RR AR Eee RE 5 Preparing the Reagent eer ete ergo eate c Dx Ee dae nre ca ex a Scu dg dees 6 Aliguoting REAGENTS NN 6 3 Optimization Setup OPTIMIZATION ASSAY 7 PASSUMMPUOMS ics ee de Ee Ee 7 Cell and Cartridge Preparation eeeeeseseesseseeseseeeeene eene nennen enne n
33. pounds in succession that shift the bioenergetic profile of the cell Figure 1 1 One group will serve as the control with running media added as control compounds The first injection is Oligomycin ATP Coupler Oligomycin inhibits ATP synthesis by blocking the proton channel of the F portion ATP synthase Complex V In mitochondrial research it is used to prevent state 3 phosphorylating respiration Within cells it can be used to distinguish the percentage of oxygen consumption devoted to ATP synthesis and the percentage of oxygen consumption required to overcome the natural proton leak across the inner mitochondrial membrane The second injection is FCCP ETC Accelerator FCCP Carbonyl cyanide p trifluoromethoxyphenylhydrazone is an ionophore that is a mobile ion carrier FCCP is an uncoupling agent because it disrupts ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase Complex V The collapse of the mitochondrial membrane potential leads to a rapid consumption of energy and oxygen without the generation of ATP In this case both OCR and ECAR increase OCR due to uncoupling and ECAR due to the cells attempting to maintain their energy balance by using glycolysis to generate ATP Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions FCCP treatment can be used to calculate the spare respiratory capacity of
34. s can be designed to obtain information on a wide variety of other processes within the mitochondria such as substrate transport dehydrogenase activity electron transport through individual complexes ATP synthase activity and proton leak Incubation conditions have to be designed such that the process to be investigated exerts significant control over the overall rate of electron transport In summary when working with intact cells or tissues one should not assume that oxygen uptake is due to mitochondria only since cells possess a variety of oxygenases Therefore when concluding a cell respiration experiment it is advisable to determine residual respiration in the presence of electron transport inhibitors such as Rotenone or Antimycin A so as to optimize your assay results SISAJEUY 7 EI oO 7 4 oO 7 Seahorse Bioscience 5 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions This page intentionally left blank sis jeuy 7 EI oO 7 oO 7 Seahorse Bioscience 34 Index A Analyzing Inhibitory response for ATP Coupler and Mito Inhibitors A and B 16 Peak response for the ETC accelerator 13 ANUMVCIN E 2 3 Assay kit Oovervlew ese sseseennnneeeeano 1 Assumptions W lle EE 7 Stress Test certet ets 19 ATP Co pler session eet trn nne 1 3 5 Coupler response esses 27 31 WII E 28
35. s possible to quantify the most important bioenergetic diagnostics of the mitochondria within an intact cell The proton current generated by basal respiration supplies the ATP synthase and the proton leak An approximate measure of the mitochondrial ATP synthesis in the basal state can be obtained from the decrease in respiration by inhibiting the ATP synthase with Oligomycin the residual respiration being ascribed to the proton leak In practice since ATP synthase inhibition results in a slight mitochondrial hyperpolarization and the proton leak is voltage dependent this approach can underestimate the ATP synthesis and exaggerate the proton leak in the basal state Seahorse Bioscience e SISAJEUY 7 OD RK 7 oO 7 Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions The addition of Oligomycin shifts ATP production to glycolysis To supply a cell with ATP at a rate comparable to that found during aerobic glycolysis requires that the pathway accelerate to more than tenfold While most O cell lines have sufficient glycolytic capacity in some cells this may not be the case with the result that Oligomycin may induce an ATP crisis s Basal Respiration The basal respiration is the baseline oxygen consumption reading per well before compounds are injected The basal reading is determined by taking the 3rd basal measurement the last measurement before 3 compound
36. ser Manual XF24 Instructions Analyzing the Inhibitory Response for the ATP Coupler and Mito Inhibitor A and Mito Inhibitor B When the ATP Coupler or Mito Inhibitor A or Mito Inhibitor B are injected into the wells the OCR response should decline in a dose dependent manner These compounds should be run separately to determine the proper concentration for inhibition for each compound As the responses are similar for these three compounds the technique for analyzing the data is the same 1 When the optimization experiment is complete open the excel data output file and choose the worksheet tab labeled Mito Stress Test Optimization see Figure 4 5 ATP Coupler Dose Response ATP Coupler OCR pMoles min 0 10 206 0 50 149 075 DI 100 83 E150 z e 8 040 ATP Coupler uM 100 10 00 CH H Data Viewer _ Mito Stress Test Optimization Assay Configuration Levels Rate Data Rate Data Pate View TIN Figure 4 5 Screen shot of the Mito Stress Test Optimization excel output for the ATP Coupler The graph on the left shows a dose response for the compound tested The table in the middle shows the data used to generate the graph compound concentration and OCR response The software will calculate the inhibitory response from the graph in the green box labeled Concentration at Dip The user can change this concentration as needed to determine the proper vol
37. spiration is now controlled by electron transport chain activity and or substrate delivery The increased respiratory capacity above basal respiration provides the spare respiratory capacity Finally electron transport chain inhibitors are added any residual respiration is non mitochondrial and needs to be subtracted from the other rates XF Cell Mito Stress Test Kit Contents The XF Cell Mito Stress Test Kit contains enough reagents to run a mitochondrial profile experiment for 6 full XF24 plates The kit contains the following reagents Reagent Name Chemical Name ATP Coupler Oligomycin ETC Accelerator FCCP Mito Inhibitor A Antimycin A Mito Inhibitor B Rotenone DMSO Dimethyl Sulfoxide Other Requirements The following is a list of the required equipment and supplies not included in the XF Cell Mito Stress Test Kit e XF24 Extracellular Flux Analyzer e Seahorse assay media or equivalent supplemented with fresh sodium pyruvate glutamine and glucose See the XF assay media product insert for more details e XF24 FluxPak including cell plates calibrant and cartridges Serial dilution vessel 24 well plate or eppendorf tubes Incubator set to 37 C without CO9 e Standard cell culture equipment Class Il Biological Safety Cabinet e P10 P1000 pipettes e Cell culture incubator Bench top Vortex Bench top mini centrifuge Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF2
38. tions 5 Dothe following a Inthe Cell seeding box enter the number of cells seeded per well 2 2 b ln the Average basal OCR box enter the average OCR NOTE The average basal OCR value for the cell in question should have been determined prior to running the optimization assay when optimizing for cell seeding concentration c Enter the final working concentration for each reagent that will be injected J 6 Click the Next button E Q o The group info screen appears 3 v H Leurs d Seahorse Oe Buren fils LUR sidoj LIE ATE LX XF Cell Mito Stress Test Kit bog Ed Figure 5 3 Group Info D X Screen o Wells in group wi 3 c N oe Group color o E 3 lt N SS o o 1 o Reset Tab 7 Assign a group to the unassigned wells by choosing a color and a name and then clicking on the appropriate wells NOTE The groups are defined as different treatments before the XF Cell Mito Stress Test is run All wells will get the same compound injections when the stress test is run o d in 7 in 77 8 Click the Next button sis jeuy 1S9J SS9J1S Seahorse Bioscience ai Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions The Stress Test Injection Layout screen appears This screen shows the injection layout for the Stress Test cartridge Oo l Guest Seahorse XE24 Corsent die LAOS A
39. tor Response The maximum rate after ETC Accelerator injection ATP Coupler Response The minimum rate after ATP Coupler injection NOTE For the calculated metrics Spare Respiratory Capacity and Coupling Efficiency the minimum Mito Inhibitor Response is subtracted from the injection responses to account for non mitochondrial respiration Spare Respiratory Capacity Spare respiratory capacity is calculated by noting the OCR response to the ETC Accelerator and dividing that number by the basal respiration and multipying by 100 to get a percentage The non mitochondrial respiration is subtracted from both of these values This is the true stress test measure of the assay as it provides the user an idea of a cells maximum ATP production therefore cells with a higher capacity have a greater ability to respond to stress Gi xr24 stre Ou A 8 1 Mito Stress Test Output Figure 6 2 2 3 Spare Respiratory Capacity Spare Average StdDev cv Respiratory 257 3 10 6 153 1 8 8 e Capacity 295078 sa L These data are 308 296 14 596 d 171 2 21 3 presented as a 237 0 13 3 56 response from basal readings with non Spare Respiratory Capacity mitochondrial respiration subtracted out Data Viewer Mito Stress Test Output Assay Configuration Levels Rate Data Seahorse Bioscience S SISAJEUY 7 OD RK 7 o 7 Seahorse BioScience XF
40. ume for each well is 500 ul 4 Place the cell plate in a 37 C incubator with no CO for one hour prior to the assay NOTE Seahorse recommends medium changes using the XF Prep Station to insure accurate final volumes When using the XF Prep Station the final volume should be set to 500 ul Seahorse Bioscience Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Setting Up the XF Cell Mito Stress Test Software 1 Open the XF software Figure 5 1 Seahorse Mito Kit Screen Standard Seahorse Apps XF Cell Stress Test Kit v Start App About this screen 2 ln the Seahorse Apps drop down menu choose XF Cell Mito Stress Test Kit 3 Click the Start App button 4 Click the Run Stress Test button The XF Cell Stress Test Setup screen appears ust Seahorse E24 Current Filey BOTA ASSAY TEMPLATE XF24 XF Cell Mito Stress Test Kit KAL Figure 5 2 XF Cell Stress Test Setup Screen XF Cell Stress Test Setup Working Port Concentration uM 7 Cell seeding A ATP Coupler ED oo A Average basal OCR B ETC Accelerator 25 1000 NNN Mito Inhibitors A ej Mito Inhibitors B O e Reset Tab Seahorse Bioscience i Maan EISES s u e y uoneziundo sis jeuy uoneziundo sis jeuy 1S9J SS9J1S o EI o KA 7 o o Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instruc
41. ume of compound stock to add to 5 ml Seahorse Assay Medium for the Cell Mito Stress Test 2 The software automatically produces a dose response curve for the compound tested This curve is shown with compound concentration on the x axis and OCR on the y axis 3 The software will automatically suggest a concentration at the inhibitory concentration of the curve but it is very important for the user to check that this is the true maximal inhibition and change accordingly 4 When the inhibitory concentration is determined type it into the field labeled Concentration at Dip 5 The software will automatically calculate the volume of 2 5 mM stock compound needed to make 5 ml of the final injection the red box Write this volume down in your notebook and use this to prepare injections for the mito stress test Seahorse Bioscience 16 gt 5 gt lt 9 o uoneziundo Seahorse BioScience XF Cell Mito Stress Test Kit User Manual XF24 Instructions Determining the Inhibitory Concentration In some situations the inhibitory response concentration is not always obvious The following presents a few examples of data from an optimization experiment and how Seahorse recommends the inhibitory concentration be chosen ATP Coupler Dose Response Figure 4 6 In this example the answer if obvious the OCR values falls as ATP Coupler concentration increases and flattens out once the nadir is reached Here the user shoul
42. unction in areas such as Obesity diabetes aging cancer cardiovascular function and safety toxicity Cellular metabolism is the process of substrate uptake such as oxygen glucose fatty acids and glutamine and the subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions These intracellular biochemical reactions result in the production of ATP and the release of heat and chemical byproducts such as lactate and CO into the extracellular environment Valuable insight into the physiological state of cells and the alteration of the state of those cells can be gained through measuring the rate of oxygen consumed by the cells an indicator of mitochondrial respiration the Oxygen Consumption Rate OCR Cells also generate ATP through glycolysis the conversion of glucose to lactate independent of oxygen The measurement of lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells causing acidification of the medium provides the Extracellular Acidification Rate ECAR This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both the physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and or insults The cells are metabolically perturbed by the addition of three different com
Download Pdf Manuals
Related Search